Secondary Metabolite Niches: Whole Genome Functional Analysis of Non-Ribosomal Peptide Synthetases

该项目的目标是进行一套详尽的表征Cochliobolus heterostrophus非核糖体肽合成酶（NRP）基因，和小分子肽产生的酶，他们编码，以确定什么肽次级代谢产物做（和）真菌细胞。非核糖体肽合成酶（NRPS）是通过一种机制产生小的非核糖体肽（NRP）的多模块酶，该机制独立于最已知用于蛋白质合成的细胞器核糖体。迄今为止，NRPS方法的肽生物合成已被描述为丝状子囊菌（和在有限的程度上，为担子菌真菌）和细菌，只有。可能的NRP产物的多样性可能是无限的，因为NRP可以由D-和L-氨基酸、蛋白质和非蛋白质氨基酸、羟基酸、鸟氨酸和其他不寻常的成分组成。此外，NRP可以是直链、环状或支链环状的，并且可以通过糖基化、N-甲基化或酰化修饰。除了结构多样性，NRPs还具有极其广泛的生物活性。然而，尽管事实上，肽产物的活性，关于与其他生物体的相互作用，是有据可查的，当然是显著的，表明这是它们的主要功能可能是不正确的。例如，12 C中只有一个。Heterostrophus peptide基因是对宿主玉米的毒力所必需的。事实上，这些小肽对生产真菌的生理意义在很大程度上是未知的。该项目的基础是假设NRPS酶是基础代谢和专门环境小生境的小分子提供者。这个项目的目标是创建一个C库。heterostrophus β-缺失菌株，其将用于解析小分子非核糖体肽在真菌细胞生物学中发挥的作用。具体而言，实验设计为：A.创建多个β-内酰胺酶基因缺失菌株。B。创建其中每一个p2p基因都过表达的菌株。C.进行一系列表型分析以探索功能。D.确定在何种条件下表达的基因。E.对野生型和具有感兴趣表型的选定突变株进行化学分析，以鉴定NRP结构。这项研究将允许一个受欢迎的本科生物学课程的可选研究模块的发展。这个外展合作计划将通过交织和传播对地球上第二大生物群真菌的兴奋来揭露和帮助学生进入科学管道。感兴趣的学生将被邀请参加一两次会议，在会上他们将被介绍NRPS的概念。然后，学生将被分配协助并最终负责表型测定。学生还将面临挑战，使用当代分子遗传学/基因组学工具来回答有关重要真菌代谢产物的起源和功能的问题，包括它们在农业中的应用及其社会影响。

The goal of this project is to undertake an exhaustive characterization of the set of Cochliobolus heterostrophus non-ribosomal peptide synthetase (NPS) genes, and the small molecule peptides produced by the enzymes they encode, to ascertain what peptide secondary metabolite products are doing in (and for) the fungal cell. Non-ribosomal peptide synthetases (NRPSs) are multimodular enzymes that make small non-ribosomal peptides (NRPs) through a mechanism, independent of organelles best known for protein synthesis, the ribosomes. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycete fungi (and to a limited extent, for basidiomycete fungi) and for bacteria, only. The diversity of possible NRP products is potentially limitless as NRPs can be composed of D- and L-amino acids, protein and non-protein amino acids, hydroxy acids, ornithine, and other unusual constituents. Furthermore, NRPs can be linear, cyclic, or branched cyclic, and may be modified by glycosylation, N-methylation or acylation. In addition to structural diversity, NRPs have an astonishingly broad spectrum of biological activities. However, despite the fact that activities of the peptide products, with respect to interactions with other organisms, are well documented, and certainly remarkable, to suggest that this is their primary function is likely incorrect. For example, only one of the 12 C. heterostrophus NPS genes is required for virulence on the host, maize. In fact, the physiological significance of these small peptides to the producing fungi is largely unknown. The foundation of this project is the hypothesis that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches. The goals of this project are to create a library of C. heterostrophus NPS- deletion strains that will be used to resolve roles that small molecule, non-ribosomal peptides play in the biology of the fungal cell. Specifically, experiments are designed to: A. Create multiple NPS gene-deletion strains. B. Create strains in which each NPS gene is overexpressed. C. Perform a series of phenotypic assays to explore function. D. Determine conditions under which NPS genes are expressed. E. Perform chemical analyses on wild-type and selected mutant strains with interesting phenotypes, to identify NRP structure. This research will allow the development of an optional research module for a popular undergraduate biology course. This outreach collaboration program will expose and help move students into the science pipeline by interweaving and disseminating excitement about the second largest group of organisms on earth, the fungi. Interested students will be invited to one or two sessions where they will be introduced to the concept of NRPSs. Then, students will be assigned to assist with and eventually take charge of phenotypic assays. Students will also be challenged to use contemporary molecular genetics/genomics tools to answer questions about the genesis and function of important fungal metabolites including their applications in agriculture and their social implications.